title: Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation.
abstract: BACKGROUND
  Expression of Y-box binding protein-1 (YB-1) is associated with tumor progression and drug resistance. Phosphorylation of YB-1 at serine residue 102 (S102) in response to growth factors is required for its transcriptional activity and is thought to be regulated by cytoplasmic signaling phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. These pathways can be activated by growth factors and by exposure to ionizing radiation (IR). So far, however, no studies have been conducted on IR-induced YB-1 phosphorylation.
  
  
  METHODS
  IR-induced YB-1 phosphorylation in K-RAS wild-type (K-RASwt) and K-RAS-mutated (K-RASmt) breast cancer cell lines was investigated. Using pharmacological inhibitors, small interfering RNA (siRNA) and plasmid-based overexpression approaches, we analyzed pathways involved in YB-1 phosphorylation by IR. Using γ-H2AX foci and standard colony formation assays, we investigated the function of YB-1 in repair of IR-induced DNA double-stranded breaks (DNA-DSB) and postirradiation survival was investigated.
  
  
  RESULTS
  The average level of phosphorylation of YB-1 in the breast cancer cell lines SKBr3, MCF-7, HBL100 and MDA-MB-231 was significantly higher than that in normal cells. Exposure to IR and stimulation with erbB1 ligands resulted in phosphorylation of YB-1 in K-RASwt SKBr3, MCF-7 and HBL100 cells, which was shown to be K-Ras-independent. In contrast, lack of YB-1 phosphorylation after stimulation with either IR or erbB1 ligands was observed in K-RASmt MDA-MB-231 cells. Similarly to MDA-MB-231 cells, YB-1 became constitutively phosphorylated in K-RASwt cells following the overexpression of mutated K-RAS, and its phosphorylation was not further enhanced by IR. Phosphorylation of YB-1 as a result of irradiation or K-RAS mutation was dependent on erbB1 and its downstream pathways, PI3K and MAPK/ERK. In K-RASmt cells K-RAS siRNA as well as YB-1 siRNA blocked repair of DNA-DSB. Likewise, YB-1 siRNA increased radiation sensitivity.
  
  
  CONCLUSIONS
  IR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-Ras or IR enhances repair of DNA-DSB and postirradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways.
authors.affiliation: Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, Eberhard Karls University Tübingen, Roentgenweg 11, D-72076 Tübingen, Germany.
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Toulany
authors.forename: Tim-Andre
authors.initials: TA
authors.lastname: Schickfluss
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Eicheler
authors.forename: Rainer
authors.initials: R
authors.lastname: Kehlbach
authors.forename: Birgit
authors.initials: B
authors.lastname: Schittek
authors.forename: H Peter
authors.initials: HP
authors.lastname: Rodemann
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism*; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair; D005260:Female; D011905:Genes, ras; D006657:Histones / Q000032:analysis; D006801:Humans; D008024:Ligands; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018739:Oncogene Proteins v-erbB / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation / Q000528:radiation effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D011836:Radiation Tolerance / Q000187:drug effects; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000528:radiation effects; D051840:Y-Box-Binding Protein 1 / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*
chemical_list: C468783:H2AX protein, human; D006657:Histones; D008024:Ligands; D018739:Oncogene Proteins v-erbB; D011838:Radiation-Sensitizing Agents; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases